一种检测β溶血性链球菌的荧光环介导等温扩增方法的建立

Establishment of a fluorescent loop-mediated isothermal amplification method for detection ofβStreptococcus hemolyticus

目的建立荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法用于β溶血性链球菌的快速检测。方法针对β溶血性链球菌spy1258基因设计4对特异性引物,对建立的LAMP体系优化其反应扩增条件,对该方法特异性和灵敏度评价,并将该方法和国标法分别应用于10份牛奶样品的检测。结果该方法特异性强,内外引物比为3:1,反应温度为63℃时达到最佳反应条件,检测灵敏度达100fg/μL,检出限低至9.8CFU/mL,且10种样品的LAMP法与传统国标法检测结果一致。结论本研究建立的荧光LAMP法具有快速、直观、灵敏度高和特异性强等优点,可应用于食品β溶血性链球菌的初步筛选。

Objective To develop a fluorescent loop mediated isothermal amplification(LAMP)method for the detection ofβStreptococcus hemolyticus.Methods Four pairs of specific primers were designed for spy1258 gene ofβStreptococcus hemolyticus.The amplification conditions of LAMP system were optimized,and the specificity and sensitivity of the method were evaluated.At the same time,this method and the traditional national standard method were used to detect 10 kinds of milk powder samples.Results The method was specific and intuitive,the best reaction conditions were obtained when the ratio of inner and outer primers was 3:1 and the reaction temperature was 63℃,the sensitivity of the method could reach 100 fg/μL,the detection limit was as low as 9.8 CFU/mL,and the results of LAMP method were consistent with those of traditional national standard method for 10 kinds of milk samples.Conclusion The fluorescent LAMP method established in this study is rapid,intuitive,sensitive and specific,and can be used for the preliminary screening ofβStreptococcus hemolyticus in food.